The present invention relates to a colorimetric method, and more particularly to a colorimetric method suited to be applied in analyzing blood sample which has an influence by chyle, hemolysis and icterus.
A recent tendency of automation in the field of biochemistry is remarkable. Various types of automatic clinical analyzers including a flow type analyzer in early time and a discrete type analyzer in modern time have been developed. Those automatic clinical analyzers have made remarkable contribution in increasing total number of test items and improving precision of measurements. However, from the viewpoint of correctness of the measurements, the presently available automatic clinical analyzers still have some problems and they are far from the requirement of users. Among others, various chromogens such as hemolysis (hemoglobin), icterus (birilbin) and chyle (turbidity) cause the loss of exactness of the measurements by the automatic clinical analyzers.
The influence by the disturbing chromogens is particularly remarkable in colorimetric method and nephelometric method using endpoint method, and it is very little in a rate method (reaction rate measurement method). However, the utilization of the rate method to all materials to be tested biochemically is principally possible but problems exist in the prices of reagents, complexity of processing and processing speed. Accordingly, in present days, very high percentage of overall tests are made by the colorimetric method.
Accordingly, if a colorimetric method (including nephelometric method) which is free from the influence by the chromogens and provides high correctness is developed, it will play an important role to the automation of the analyzer.
One method which has been heretofore been considered to be most basic one to prevent the disturbance by those chromogens is to measure test sample blank for each test sample and test item. Although there are many problems to be discussed, such as composition of reagent, basically the test sample blank is measured using a reagent which does not include certain ones of materials which are relevant to the measurement reaction, and the test sample is determined from a difference between the measurement by that reagent and the measurement by reaction solution for the test sample. In this method, it is possible to obtain correct measurement of analysis which is close to the true value and free from the influence by the disturbing material. However, when such test sample blank correction method is used in the automatic clinical analyzer, two times of measurements are required for each sample and the processing speed of the analyzer necessarily reduced to one half and increased number of reagents are required. Accordingly, this method is presently not used except certain test for special items.
Another factor which causes the loss of exactness of the measurements in the colorimetric method is air bubbles and solids. These are observed both in the colorimetric method of flow cell type and in the direct colorimetric method using a test tube.
There are more than 40 sorts of materials to be measured by the colorimetric method in the biochemical test. Heretofore, a single wavelength method or a dual wavelength method has been used in the colorimetric method. The former measures a light absorbance at a particular wavelength near an absorbance center of a reacting material corresponding to the test sample or a reaction product, and the latter measures a difference between light absorbances at a wavelength near the absorbance center and an another appropriate wavelength. However, both methods are subjected to the influence by the disturbing chromogens.